Kaposi sarcoma herpesvirus (KSHV) is an oncogenic human herpesvirus, which causes Kaposi sarcoma (KS) as well as B cell lymphomas in the absence of adequate immune control. KSHV-associated tumors are a significant cause of morbidity and mortality in transplant patients and individuals with HIV-disease. The oral cavity is an important site for KSHV replication and persistence and saliva is believed to be a critical source of person-to-person transmission. Moreover, oral manifestation of KS is associated with rapid progression and high mortality rates. KSHV-associated tumors are multi-focal and frequently polyclonal, suggesting that continued viral infection is important for disease progression. Because of technical challenges unique to KSHV molecular virology, little is known about infection of physiologically relevant cell types. The current project will leverage new molecular genetics tools to explore the fundamental virology of KSHV infection with the goal of identifying novel viral and cellular targets to prevent viral spread and KS progression within the oral cavity. During the K99 phase, we will establish systems for de novo infection of KSHV-susceptible cell types and tissues in order to explore the cell type-specific properties of infectin, persistence and spread. We will then utilize these models to explore the contribution of the viral FLICE- inhibitory protein (vFLIP) in the context of KSHV infection. In order to place these studies in the most disease-relevant context possible, we will include HIV co-infection, as appropriate, to determine fundamental interactions between these two pathogens. With critical training and career development accomplished, model systems in hand, and the vFLIP studies as proof-of-principle for the use of recombinant KSHV strains within these models, I will move forward in the R00 phase to examine the contribution of virion-associated non-structural KSHV proteins to the earliest stages of infection in relevant cell types. These packaged, non-structural proteins comprise a virion structure known as the tegument. Tegument factors affect profound alterations in host cell physiology immediately upon virus entry and studies of these factors in other herpesvirus systems have yielded important insights into viral and cellular biology. However, the composition and function of the KSHV tegument remains obscure and represents a large gap in our understanding of fundamental KSHV virology. The results of this research will provide critical information about the dynamic process of KSHV persistence in the oral cavity and provide insights into both viral and cellular mechanisms that can be exploited for new treatment paradigms. Using a broad but systematic workflow of study for multiple KSHV tegument factors, I will obtain a significant body of both publishable data and preliminary data for multiple future studies that will provide a solid foundation upon which to build an independent research career.